We determined epigenetic modification signatures and chromatin structure at high resolution across 300 Kb of human chromosome 11, including the beta-globin locus, in human K562 cells where the embryonic epsilon and fetal gamma genes are expressed and the adult beta-globin genes are silent. To investigate a correlation between epigenetic modification of these genes and their sequential activation during development, we chose an in vitro differentiation system in which human CD34+ cells are differentiated over two weeks and eventually express each of the globin genes including the adult genes. Because these cells are in limiting number we optimized a protocol for mini-chromatin immunoprecipitation using a lower cell number than is typical. The results thus far are consistent with our data in K562 cells and indicate early epigenetic modification of the locus control region enhancer before the globin genes are expressed and the progressive detection of active histone marks across the locus as the genes are activated.[unreadable] [unreadable] Histone H3 and H4 acetylation and H3 K4 di-methylation are marks of permissive chromatin generally associated with actively transcribed genes in metazoans. In contrast, H3K9 di- and tri-methylation (H3K9me2 and me3) are generally accepted to be features of silent heterochromatin. Surprisingly, we and others found H3 K9 tri-methylation associated with actively transcribed globin genes. There are at least 6 enzymes in human cells that are known to carry out H3 K9 methylation at different levels. G9A is generally associated with euchromatic lysine 9 dimethylation but has trimethylation activity in vitro. Using ChIP, we found that G9A is recruited to the human beta-globin LCR HSs and active globin gene promoters. G9A and the related methyltransferase GLP form a heteromeric complex that is essential for their activity in vivo. To determine whether G9A or GLP are responsible for the H3K9me3 we observed in actively transcribed globin genes, we knocked down their expression singly and concurrently in erythroid cells using RNAi and we are examining the consequences for globin transcription and localization of HMTase complex components. In addition, we will knockdown the K4 HMT complex component Ash2L and examine the effect on transcription and K4 and K9 methylation to determine whether K4 and K9 methylation marks are functionally related.